1. Field of the Invention
The present invention relates to a method of preventing and treating thrombosis, acute crisis in sickle cell anemia, neoplastic metastases and certain aging disorders.
2. Description of the Background Art
Arterial and venous thromboemboli cause common, serious or life-threatening disorders. Coronary arterial thrombosis leads to coronary ischemia and infarction. Thrombotic or embolic occulusions of cerebral arteries may result in ischemia or infarction of the central nervous system. Venous thrombic disorders are associated with emboli that occlude branches of the pulmonary arterial circulation and frequently complicate the course of other serious illnesses or the recovery from injury or surgery.
Therapeutic recommendations for thromboembolic disorders remain controversial and are sometimes confusing. Recent studies have provided considerable insight into the pathophysiology of these disorders. In order to understand the thromboembolic phenomena, an understanding of normal hemostasis is necessary. To summarize briefly, vascular injury initiates a sequence of events that results in the formation of a platelet-fibrin barrier to limit escape of blood. The initial vessel damage exposes sub-endothelial structures to the bloodstream, and blood platelets begin to adhere and aggregate at the site of injury due to their interaction with factor VIII-related von Willerbrand factor (vWF) and collagen. Proteins of the coagulation system are also activated to generate the enzyme thrombin. Thrombin cleaves plasma fibrinogen into fibrin monomers, which polymerize around the clumped platelets and hold them in place, forming the primary hemostatic plug. Over the following 8-24 hours, additional fibrin is laid down, forming the secondary hemostatic plug and allowing the repair of the underlying vessel wall to proceed. Subsequently, this clot is digested by the fibrinolytic enzyme, plasmin, which is activated in situ (Moake, Clinical Symposia, Volume 35, No. 3, 1, 1983).
One of the major physiologic mechanisms of platelet thrombus formation is vWF-mediated platelet aggregation. Much is known about the interaction of vWF with its major receptor, glycoprotein Ib (Bockenstedt, et al., J. Clin. Invest. 77: 743, 1986), but there are no specific chemical inhibitors of this binding in common use. Additionally, there are situations in which a drug to interrupt this association in humans may be beneficial. These includes coronary and cerebral thrombosis, thrombotic thrombocytopenic purpura and the hemolytic-uremic syndrome.
vWF is a large, multimeric plasma protein (subunit molecular weight of 2.25.times.10.sup.5 daltons) synthesized by bone marrow megakaryocytes and endothelial cells that form the lining of blood vessels. Circulating vWF is heterogeneous in size, with multimers ranging from about 4.times.10.sup.5 to millions of daltons. Large vWF multimeric forms are involved in platelet-subendotheial adhesion and in shear stress-induced platelet aggregation. Large vWF forms bind to platelet surfaces via glycoprotein molecules embedded in the cell membrane.
Human endothelial cells in culture synthesize "unusually large" vWF multimers which are larger than the largest multimeric forms found in normal human plasma ("plasma-type" vWF multimers). These unusually large vWF forms are secreted by endothelial cells into the subendothelial matrix and, under certain conditions, into the plasma. The unusually large vWF multimers are very adhesive to platelets. When endothelial cells are damaged, platelets adhere to collagen and vWF multimers, perhaps especially the unusually large forms in the exposed subendothelium.
In the arterial circulation, the platelet adhesion and aggregation that follows vascular damage may sometimes lead to arterial thrombosis. If a platelet thrombus forms and completely or substantially occludes the arterial lumen, blood flow is slowed or stopped distal to the occlusion. This diminished blood flows leads to coagulation factor activation, thrombin generation, fibrin polymer formation, and additional thrombin-induced platelet aggregation distal to the site of vessel injury. As a result, the initial thrombus, composed predominantly of platelets, extends distally as fibrin formation occurs. Arterial emboli (portions of thrombi that break away into the bloodstream) may also partially or completely occlude distal arterioles or capillaries, causing ischemia or infarction of the tissues supplied by these vessels. Emboli originating from a thrombus on an atherosclerotic cerebral artery can cause transient partial occulsion of a distal vessel (transient ischemic attack) or complete vascular occulsion and infarction of brain tissue (stroke).
Thrombotic thrombocytopenic purpura (TTP) and the hemolytic-uremic syndrome (HUS) are diseases caused by platelet aggregation in the arterial circulation. In TTP, the formation of platelet clumps occurs in the arterial system, resulting in the occlusion of some arterioles and capillaries. In HUS, the intravascular platelet clumping is confined almost exclusively to the renal arterial vessels. In both syndromes, the intravascular clumping of platelets is the cause of the thrombocytopenia. In addition, erythrocyte fragmentation occurs in both disorders because red blood cells are injured as they move through the partially occluded arterioles and capillaries.
In 1975, Kirby reported that Evans Blue, a common dye formerly used in biological laboratories for blood volume determination, interrupted the association of formaldehyde-fixed human platelets with human vWF and ristocetin, or bovine vWF alone. (Kirby, Thhrombos Diathes Haemorrh. 34: 770, 1975.) This reaction is properly termed platelet agglutination, which is the clumping of platelets induced by vWF binding to the platelet surface, mostly via glycoprotein Ib, and is not necessarily associated with a secondary platelet granule release reaction. In contrast, platelet aggregation requires functional, metabolically-active platelets. Platelet aggregation is induced by a variety of stimuli, including ADP, collagen or arachidonic acid and results in irreversible platelet shape change and granule release. Subsequently, Geratz et al. (Thrombos Haemostas 39: 411, 1978) tested 20 custom synthetic compounds with structural similarity to Evans Blue and found that minor changes in substituent groups on the phenol rings altered the platelet aggregation-inhibitory activity of these compounds and, in some cases, caused a differential change in the anti-aggregation and anti-agglutination activities. In their assay system, suramin (an antitrypanosomal drug) was a weak anti-aggregation agent.
In vitro, ristocetin (a negatively-charged antibiotic) or fluid shear stresses alter the platelet surface so that vWF multimers attach to the platelets and cause them to clump.
Unusually large vWF multimers, which resemble those synthesized by normal human endothelial cells in culture, have been found in the plasma of some patients with the chronic relapsing type of TTP. These unusually large vWF multimers are not found in normal plasma. The plasma content of unusually large vWF multimers decreases during relapses in this disorder, presumably because they have become attached to agglutinating platelets. A mechanism similar to that of ristocetin causing in vitro attachment of vWF to platelet surfaces may be present intermittently in chronic relapsing TTP. Cationic peptides or other molecules released periodically from injured tissue or phagocytic cells may induce the selective attachment of unusually large vWF multimers to circulating platelets, and thus cause the platelets to agglutinate within arterioles. In the acute, non-relapsing type of TTP, unusually large vWF multimers may enter the circulation as a result of extensive endothelial cell injury or stimulation.
Prior to the present invention the preferred therapy of TTP and HUS consists of prophylactic or therapeutic transfusion of normal plasma, which may provide both temporary additional vWF depolymerase activity and additional plasma proteins to bind and eliminate the proposed inciting agents in clinical relapses. During severe episodes, partial removal of the unusually large vWF multimers by plasmapheresis, combined with transfusion of normal plasma, is often required to control platelet agglutination.
It has been suggested that HUS may be a variant of nonrecurrent TTP with intravascular platelet clumping confined to renal vessels. Recent findings indicate that large vWF multimers may also be involved in the pathophysiology of HUS, perhaps as a result of renal endothelial cell injury (or intense stimulation). Attempts to treat TTP and HUS with drugs such as acetylsalicylic acid (ASA, or aspirin), ibuprofen-type compounds (Motrin, Advil, etc.), imidazole compounds, and dipyridamole, which either directly or indirectly suppress the release of platelet granule contents (including ADP from dense granules and PDGF from a-granules) haven been of equivocal benefit. Because blood flow slows in leg veins of patients confined to bed or in those with increased intraabdominal pressure (for example, during pregnancy) coagulation factors are more likely to be activated excessively under these hemodynamic conditions. There may also be some regional increase in vWF-mediated platelet-subendothelial interaction. The resulting venous thrombosis in an iliac or deep femoral vein may cause pain and swelling of the leg. Fragments of thrombi in pelvic, iliac, or deep femoral veins are especially likely to embolize and occlude arterial branches of the pulmonary circulation, with potentially life-threatening consequences.
Another pathological condition which involves vaso-occlusion is sickle cell anemia. Among the major manifestations of sickle cell anemia are periodic, localized, vaso-occlusive crises and chronic hemolytic anemia. Adhesion of sickle erythrocytes to the vascular endothelium has been proposed as one mechanism of vaso-occlusion. Sickle red cells adhere abnormally to cultured endothelial cell under both static and flow conditions. This increase in adhesion, when compared to normal red cells, has been related to the clinical severity of vaso-occlusive events in sickle cell disease (Hebbel, et al., New England J. Med 302: 992, 1980). Wick demonstrated that unusually large vWF multimers mediate the adhesive interactions between sickle erythrocytes and endothelial cells (Wick T. M., et al., J. Clin. Invest., 80: 905, 1987).
Interference with platelet aggregation may inhibit the capacity of neoplastic cells to metastasize (Honn, et al. Science 212: 1270, 1981). Since von Willebrand factor has been shown to be important in the implantation of circulating tumor cell-platelet clumps under certain experimental conditions, the inhibition of vWF-mediated platelet adhesion to subendothelial surfaces by the compound useful in the present invention may decrease the chance of metastasis of certain human malignancies (Marcum, et al., J. Cab. Clin. Med. 96: 1046, 1980). Since vWF-platelet interactions may play a role in the aging process (Cerami, et al., Sc. American 256(5): 90, 1987, the compounds useful in the present invention may delay certain aging phenomena.